Low doses of sex steroids, including 17-f5 estradiol (E2) and testosterone (T4), confer protection against clinical and histological experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease, multiple sclerosis (MS). We hypothesize that the CNSneuroprotective effects of both sex steroids on EAE are mediated through both immune and non-immune cells. E2 may contribute to EAE resistance by influencingthe development and function of potentially pathogenic T cells specific for myelin antigens, as well as regulatory T cells, including the newly described CD4+CD25+ Treg cells, which might modify the course of disease. Alternatively, the primary effects of E2 might be on macrophages, dendritic cells, or CNS cells. We found that E2 treatment inhibited encephalitogenic activity of myelin-specific T cells, reduced cytokines (particularly TNF-a) and chemokines/receptors in the CNS and spleen, and inhibited recruitment of inflammatory cells into the CNS. Recently, we demonstrated that ERKO mice lackingEsrl (a receptor for E2) but not BERKO mice lacking Esr2 (P receptor for E2) were refractory to E2-mediated inhibition of chronic EAE, thus implicatingEsrl in E2-mediated protection. In Aim 1, we will determine if immune or non-immune cells mediate the neuroprotective effects of E2 in chronic (C57BL/6) and relapsing (SJL) models of EAE. In Aim 2, we will pursue promising preliminary data indicating that E2 may potentiate the regulatory activity of CD4+CD25+ T cells that may also function through Esrl. T4 may also provide protection from EAE, as we have previously shown. Castration worsens EAE in our young male mouse model, while exogenous T4 treatment reduces disease. We hypothesize that T4 provides protection because it is converted to E2 via the P450 aromatase, and this effect is most pronounced in middle-aged males. Alternatively, T4 may act by direct signaling through the androgen receptor (AR). In Aim 3, we will test the hypothesis that exogenous T4 treatment provides protection against EAE through enzymatic conversion that is age dependent. Results obtained from this project will selectively identify E2-responsive cell types in EAE and determine if estrogen and testosterone protection against EAE utilize overlapping mechanisms, thus providing a direct comparison with neuroprotective mechanisms of T4 in cerebral ischemia (Project 2). Our studies may be useful in predicting how testosterones might benefit middle- aged patients with MS.